Retroviral vectors promote the transfer of genes into a variety of cell types from many animal species. Retroviral vectors are among the primary vehicles used for gene transfer into human somatic cells because of their ability to transfer genes efficiently into cells that are difficult to transfect by other methods.
A critical element in the production of the components to carry out retroviral mediated gene transfer is the cell that generates the retroviral particles carrying the gene to be transferred. These cells are called "packaging cells" because they "package" the retroviral vector which carries the gene of interest into a delivery vehicle, the retroviral particles. Packaging cell lines are designed to synthesize all retroviral proteins required for assembly of high-titer infectious virus, but should not produce any replication-competent virus. Thus, the retroviral vector consists of DNA sequences intended for transfer flanked by signals present at the ends of the retroviral genome, and the packaging cells are designed to produce all of the retroviral proteins and promote "packaging" of the retroviral RNA into virions. Retroviral vectors produced by using packaging cells can thus infect cells but cannot replicate further.
Retrovirus packaging cells provide useful tools for a variety of gene transfer applications. However, not all cell types can be efficiently infected by using the available packaging cell lines. The range of cells that are infectable by a retroviral particle is primarily determined by the envelope proteins of the virus and the presence of appropriate receptors for this protein on the surface of target cells. For example, viruses that infect human cells can be separated into eight groups based on the use of different receptors for cell entry.
Recent improvements include the design of packaging cells to produce vectors having a vesicular stomatitis virus G protein coat for expanded host range (Burns et al., Proc. Natl. Acad. Sci. U.S.A. 90:8033-8037 (1993); Lin et al., Science 265:666-669 (1994)), vectors that are resistant to human serum (Cosset et al., (1995)), and vectors that target to new cell-surface proteins (Kasahara et al., Science 1373-1376 (1994); Somia et al., Proc. Natl. Acad. Sci. U.S.A. 92:7570-7574 (1995)). However, there is still room for improvement to increase the efficiency and range of cell types that can be transduced using existing retroviral vectors. For example, treatment of genetic and acquired disease in humans would be greatly facilitated by the ability to transfer genes into hematopoietic stem cells, but transduction of these cells in large animals and humans remains low.
Packaging cells which produce amphotropic retrovirus (retroviruses which can infect cells from many species) were developed over ten years ago and are still commonly used because of the wide range of cell types from different species, including humans, that these vectors can transduce. (Cone et al., Proc. Natl. Acad. Sci. U.S.A. 81:6349-6353 (1984); Miller et al., Mol. Cell. Biol. 6:2895-2902 (1986); Miller et al., Mol. Cell. Biol. 5:431-437 (1985); and Sorge et al., Mol. Cell. Biol. 4:1730-1737 (1984)). More recently, packaging cells have been developed based on gibbon ape leukemia virus (GALV) (Miller et al., J. Virol. 65:2220-2224 (1991)) that produce vectors that use a different receptor than the prior known amphotrophic retroviruses for cell entry, and are capable of transducing myeloid, lymphoid, and airway epithelial cells at higher rates than amphotropic vectors do (Bauer et al., Blood 86:2379-2387 (1995); Bayle et al., Hum. Gene Ther. 4:161-170; Bunnell et al., Proc. Natl. Acad. Sci. U.S.A. 92:7739-7743 (1995); von Kalle et al., Blood 84:2890-2897).
The GALV and amphotropic retrovirus receptors are related phosphate transport proteins that exhibit wide, but different, patterns of tissue specific expression (Kavanaugh et al., Proc. Natl. Acad. Sci. U.S.A. 91:7071-7075 (1994)). The GALV receptor Glvr-1 (Pit-1) is most highly expressed in bone marrow, while the amphotropic receptor Ram-1 (Pit-2) is most highly expressed in the heart. It has been shown that 10A1 murine leukemia virus can use either mouse or human Glvr-1 or rat or human Ram-1 for cell entry (Miller et al., J. Virol. 68:8270-8276 (1994)).
What is needed in the art is a retroviral packaging system that offers advantages over currently available viral packaging systems and that is useful in a wide variety of gene transfer applications. The present invention addresses this and other related needs.